Transglutaminases are a calcium dependent family of enzymes responsible for post-translational crosslinking of proteins. The crosslinked protein products are large chemically, enzymatically, and mechanically resistant polymers with a variety of functions ranging from wound healing to skin formation (Greenberg et al., FASEB J. 5: 3071-3077 (1991)).
To date, there are several human transglutaminases which are produced from different genes. The catalytic subunit of blood-clotting factor XIIIa, or plasma transglutaminase, is 80 kDa (Ichinose et al., Proc. Natl. Acad. Sci. USA 85: 5829-5833 (1988)), the epidermal keratinocyte transglutaminase is 106 kDa (Phillips et al., Proc. Natl. Acad. Sci. 87: 933-9337 (1990)), the epidermal and hair follicle transglutaminases are 77 kDa (Kim et al., J. Biol. Chem. 268: 12682-12690 (1993)), the prostate transglutaminase is 78 kDa (Grant et al., Biochem. Biophys. Res. Comm. 203: 117-1123 (1994)), and the cellular transglutaminase is about 80 kDa (Gentile et al., J.Biol.Chem. 266: 478-483 (1991)).
Molecular cloning of cDNA for different forms of the cellular transglutaminase were reported (Gentile et al., J. Biol. Chem. 266:478-483 (1991), and a patent application number WO 92/12238), Fraij et al., J. Biol. Chem. 267: 22616-22673 (1992), and U.S. Pat. No. 5,726,051, Fraij and Gonzales, Biochim. Biophys. Acta. 1306: 63-74 (1996)). The complete organization and structure of a human cellular transglutaminase gene was recently published (Fraij and Gonzales, Biochim. Biophys. Acta. In Press (1997)).
Cellular transglutaminase is one of the most extensively studied of the transglutaminases and its crosslinking activities have been reported to be involved in skin wound healing, adhesive strength at the cartilage-cartilage interface and in the gastric mucosal injury healing (Raghunatg et al., J. Clin. Invest. 98: 1174-1184 (1996), Jurgensen et al., J. Bone and Joint Surg. 79-A: 185-193 (1997), Wang et al., Gastroenterology 104: 65-74 (1993)). Cellular transglutaminase crosslinking activities have been reported to be involved in the stabilization of apoptotic bodies (Piacentini et al., Eur. J. Cell Biol. 54: 246-254 (1991)), and to function in cellular signal transfer as a GTP receptor coupling protein (Nakaoka et al., Science 264: 1593-1596 (1994)).
Transglutaminase homologue was found recently to bind and hydrolyze GTP several fold more than the cellular transglutaminase, activities which may be related to the events of cell signaling (Fraij, Biochem. Biophys. Res. Comm. 218: 45-49 (1996)). Transglutaminase crosslinking reactions are known to occur early in regulation of receptor/membrane functions. It is evident from the patent literature that numerous concepts have been tested and are believed to have a future in the industry, however this will very much depend upon the availability and costs of the transglutaminase.
Cellular transglutaminase when compared to the other known transglutaminases, except for cellular transglutaminase, all were reported to exist as zymogen and require a limited proteolysis for full enzyme activity (Greenberg et al., FASEB J. 5: 3071-3077 (1991)). Since its discovery in 1954, the 80 kDa form of cellular transglutaminase is regarded as the active enzyme and often cited as the enzyme of the family that does not require proteolytic activation.
Transglutaminases have been used for crosslinking purposes in a variety of fields. Currently, Factor XIIIa purified from human plasma and placenta are used for intravenous injections to treat chronic venous ulcerations and as a fibrin sealant for surgery purposes. Microbial transglutaminases have found use in food industry, and since 1993 have been commercially available in Japan.
Human Factor XIIIa requires some form of activation to become catalytically active, and as each transglutaminase has certain substrates, their activities may be limited in certain applications. Therefore, what is needed in the art are methods for producing by recombinant means human transglutaminase which is catalytically active and does not require activation and does not require testing for HIV and Hepatitis viruses.